Peet R C, Lindgren P B, Willis D K, Panopoulos N J
J Bacteriol. 1986 Jun;166(3):1096-105. doi: 10.1128/jb.166.3.1096-1105.1986.
Genes involved in the production of phaseolotoxin by Pseudomonas syringae pv. "phaseolicola" NPS3121 were identified by Tn5 mutagenesis and cosmid cloning. A total of 5,180 kanamycin-resistant colonies were screened for the loss of phaseolotoxin production by a microbiological assay. Six independent, prototrophic, Tox- mutants were isolated that had Tn5 insertions in five different EcoRI fragments. All six mutants had Tn5 inserted in the same KpnI fragment, which had a length of ca. 28 kilobases including Tn5. The mutants produced residual toxin in vitro. An EcoRI fragment containing Tn5 and flanking sequences from mutant NPS4336 was cloned and used to probe a wild-type genomic library by colony hybridization. Seven recombinant plasmids showing homology to this probe were identified. Each Tox- mutant was restored in OCTase-specific toxin production by two or more of the recombinant plasmids. The data suggest that at least some of the genes involved in phaseolotoxin production were clustered in a large KpnI fragment. No homology was detected between the Tn5 target fragment cloned from mutant NPS4336 and the total genomic DNA from closely or distantly related bacteria that do not produce phaseolotoxin.
通过Tn5诱变和黏粒克隆鉴定了丁香假单胞菌菜豆致病变种NPS3121中与菜豆毒素产生相关的基因。通过微生物学测定法对总共5180个卡那霉素抗性菌落进行筛选，以检测菜豆毒素产生的丧失情况。分离出6个独立的、原养型的Tox-突变体，它们在5个不同的EcoRI片段中存在Tn5插入。所有6个突变体的Tn5均插入到同一个KpnI片段中，该片段长度约为28千碱基，包括Tn5。这些突变体在体外产生残留毒素。克隆了一个包含来自突变体NPS4336的Tn5和侧翼序列的EcoRI片段，并通过菌落杂交用于探测野生型基因组文库。鉴定出7个与该探针具有同源性的重组质粒。每个Tox-突变体通过两个或更多的重组质粒恢复了OCTase特异性毒素的产生。数据表明，至少一些与菜豆毒素产生相关的基因聚集在一个大的KpnI片段中。从突变体NPS4336克隆的Tn5靶片段与不产生菜豆毒素的近缘或远缘细菌的总基因组DNA之间未检测到同源性。
